In the previously review period we completed a Phase I and II clinical trial targeting telomerase in non-small cell lung cancer (NSCLC). The results of these trials validated modulating telomere biology as an important target in lung cancer. NSCLC patients with short telomeres have poorer progression free and overall survival compared to patients with longer telomeres. In our completed Phase II trial we determined that the patients with the shortest telomeres trended to having better overall responses to our telomerase inhibitor. These results have encouraged us to pursue other novel mechanisms to modulate telomere biology and this is the focus of this renewal project. We also have been conducting preclinical studies on cancer stem cell biomarkers in NSCLC. We have identified ALDH1A1 and ALDH1A3 as candidate lung cancer stem cell biomarkers whose expression correlates with a poor prognosis in lung cancer patients with resected Stage I disease. These putative cancer stem cell populations are telomerase positive with activated Wnt and Hh activated pathways and we have experimental support for the hypothesize that our telomerase inhibitors not only target the bulk of the lung cancer cells but also the stem cell pool. We also have specific inhibitors against ALDH and will test these for reducing clonogenicity and tumorigenicity in direct (never in cell culture) xenografts and in ROCK inhibitor established primary lung cancers. We have a strong commitment and proven track record of being able to advance telomerase inhibitors to clinical trials. We will now progress our patented telomerase inhibitors (telomere modulators) and stem cell candidate therapies. This will include a WNT inhibitor, IWR-1 that targets tankyrase (PARP5A), a telomere associated protein. We will also advance 6-thio-2'deoxyguanosine (6-thio-dG) that is a nucleoside-based telomerase substrate. Both 6-thio-dG and IWR-1 treatments show increased specificity to telomerase expressing cancer cells over normal telomerase silent cells. Both lead to telomere shortening and reduced in vitro and in vivo cell growth. Our long-term goal is to maximize telomere shortening or uncapping and to determine the most effective way to use these SPORE developed inhibitors clinically in patients with NSCLC.